Use of seed flour as soil pesticide

ABSTRACT

Use of vegetable seed flour, in particular Brassicaceae flour, as a cytotoxic agent with improving action.

TECHNICAL FIELD

The present invention concerns a use of seed flour as a soil improvingagent, as a cytotoxic agent for soil pathogens and parasites, and as afungitoxic agent for soil fungal pathogens.

BACKGROUND ART

In the agricultural sector methyl bromide is commonly used to limit thegrowth of fungal pathogens, nematodes, wireworms, insects, bacteria andweeds. However, methyl bromide has a relatively high environmentalimpact: reacting with the ozone present in the upper layer of theatmosphere, it transforms it into Br₂O, contributing to destruction ofthe ozone.

Recently, to replace methyl bromide, molecules of vegetable origin withcytotoxic activity for soil pathogens and parasites have been proposed.Among these molecules, derivatives of glucosinolates (GLs) generated byhydrolysis catalysed by the enzyme myrosinase (MYR) or by thermolysishave been proposed. The GLs are anionic thioglucosidic compounds presentin variable quantities and ratios in a number of organisms and vegetabletissues. The molecular structure of the approximately 120 GLs currentlyisolated and characterised consists of a common functional group and alateral chain which can be of aliphatic, aromatic or heteroaromatictype.

The GLs, in the presence of the enzyme MYR (thioglucosideglucohydrolase, EC 3.2.3.1), are hydrolysed with formation ofβ-D-glucose, sulphate ion and a number of derivatives such asisothiocyanates, nitrites or thiocyanates (see, for example, Tookey H.L., Van Etten C. H., Daxembichler M. E., Toxic Constituent of PlantFoodstuffs, edited by I. E. Liner, 1980, II edition, 4, 103-142).

Some of the above-mentioned derivatives of the GLs, in particular theisothiocyanates, have shown in vitro cytotoxic properties towardsnematodes (see Lazzeri L., Tacconi R., Palmieri S., In Vitro Activity ofSome Glucosinolates and Their Reaction Products toward a Population ofthe Nematode Heterodera schachtii, J. Agric. and Food Chem. 1993, 41,825-829) and plant fungal pathogens present in the soil (see, forexample, Manici L. M., Leoni O., Lazzeri L., Galletti S., Palmieri S.,Fungitoxic Activity of Some Glucosinolate Enzyme Derived ProductsAgainst the Main Soil-Borne Pathogens, Pesticide Science, 1999, 55,486-488; and Manici L. M., Lazzeri L., Palmieri S., In Vitro FungitoxicActivity of Some Glucosinolates and their Enzyme-derived Products towardPlant Pathogenic Fungi, J. Agric. and Food Chem. 1997, 45, 2768-2773).

To obtain and use the above-mentioned derivatives of the GLs, extractionand/or production procedures are necessary, which are relatively complexand costly and can also cause environmental problems (for exampledisposal of the by-products and/or the solvents used). Furthermore,generally some important derivatives of the GLs (for exampleallylisothiocyanate) are relatively difficult to handle as they have arelatively high volatility. To overcome these negative aspects thetechnique of green manure of plants containing both GLs and the enzymeMYR (i.e. containing the GLs-MYR system) has been proposed.

The green manure technique of plants containing the GLs-MYR system hassome negative aspects, however, typical of the green manure technique ingeneral. In particular, the green manure technique requires cultivationoperations that determine relatively high labour costs and causes theloss of at least one growing cycle, with consequent damage for thefarmer due to loss of income.

DISCLOSURE OF INVENTION

The aim of the present invention is to provide a use of a seed flour asa soil improver in order to reduce the above-mentioned disadvantages ina relatively simple and inexpensive way.

The present invention provides a use of a seed flour containing at leastone glucosinolate and at least one enzyme chosen from the groupconsisting of:

-   -   glucosidasic enzymes, and    -   thioglucosidasic enzymes;

as a soil improver.

A further aim of the present invention is to provide a use of a seedflour as a cytotxic agent for soil pathogens and parasites.

The present invention provides a use of a seed flour containing at leastone glucosinolate and at least one enzyme chosen from the groupconsisting of:

-   -   glucosidasic enzymes, and    -   thioglucosidasic enzymes;

as a cytotoxic agent for soil pathogens and soil parasites.

A further aim of the present invention is to provide a use of a seedflour as a fungitoxic agent.

The present invention provides a use of a seed flour containing at leastone GL and at least one enzyme chosen from the group consisting of:

-   -   glucosidasic enzymes, and    -   thioglucosidasic enzymes;

as a fungitoxic agent for fungal pathogens of the soil.

Further characteristics of the present invention will be betterclarified by the following non-restrictive description.

BEST MODE FOR CARRYING OUT THE INVENTION

Seeds containing GLs and at least one glucosidasic or thioglucosidasicenzyme are ground in order to obtain a fine uniform flour. Preferablythe seeds contain the enzyme MYR, more preferably they belong to plantsin the order of the Capparales and to the families of the Akaniaceae,Bataceae, and even more preferably they are seeds of Brassicaceae,Capparaceae, Caricaceae, Gyrostemonaceae, Limnanthaceae, Moringaceae,Pentadiplantdraceae, Koeberliniaceae, Resedaceae, Salvodoraceae,Tropeolaceae and/or Toviaraceae.

Preferably the seeds used are seeds of Brassicaceae and/or Capparaceae,in particular they are seeds of Brassica carinata A. B., seeds of ErucaSativa M., and/or seeds of Barbarea verna. Furthermore, it is importantto point out that it is possible to identify other vegetable varieties,the seeds of which contain GLs and a glucosidasic or thioglucosidasicenzyme, analysing the seeds by means of known methods.

The seed flour thus obtained has a variable content of GLs, glucosidasicor thioglucosidasic enzyme, proteinic material and oil according to thevegetable varieties and/or the ambient growing conditions. Furthermore,it is important to underline that when the vegetable varieties usedvary, the related quantities of the different types of GLs contained inthe seeds can also vary.

For each vegetable variety the best growing techniques can be identified(i.e. by optimising, for example, the sowing period, density, type ofsoil and/or fertilising agents) in order to obtain a higher content ofGLs and glucosidasic or thioglucosidasic enzyme in the seeds.

It should also be noted that, preferably, to further increase thecontent of GLs and glucosidasic or thioglucosidasic enzyme, theabove-mentioned seed flour is de-oiled, in other words it is treated inorder to extract at least part of the oil contained in it. Theextraction is performed preferably at a relatively low temperature,preferably below 75° C., in particular at ambient temperature, so thatthe glucosidasic or thioglucosidasic enzyme is not thermallydeteriorated and, also after the oil extraction phase, it is still ableto perform its enzymatic activity. In this regard it is important tounderline that the oil extracted can be used, for example, as arenewable and ecologically sustainable lubricant or hydraulic fluid.

The flour, prepared in the form of pellets, can be distributed over thesoil relatively easily and at a relatively low cost. In the presence ofwater, derivatives of the GLs generated by hydrolysis catalysed by theglucosidasic or thioglucosidasic enzyme develop in the soil. In thisregard, it is important to underline that in order to hydrolyse the GLs,water is required, which is a reagent essential for triggering anddeveloping the biochemical reaction.

For this reason, the non-hydrated flour has a relatively high stability,can be fairly easily transported, stored for relatively long periods andused at any time, regardless of the ambient conditions.

Once the flour has been distributed on the soil and hydrated, thederivatives of the GLs generated by enzymatic hydrolysis are able toperform their cytotoxic action even though the flour containsapproximately 50% protein. Therefore, although it may be feared that theisocyanates generated by hydrolysis of the GLs, catalysed by theglucosidasic enzymes, or more specifically by the thioglucosidasicenzyme MYR, may spontaneously interact with the free NH2 groups of thelysines and arginines contained in the proteins, to produce adducts of adifferent type that can be referred to derivatives of thiourea, thehydrolysis products of the free GLs are surprisingly still able toperform a relatively high improving action in the soil. The hydrolysisproducts of the GLs perform a fungitoxic action, in particular on fungalpathogens of the soil belonging to different taxonomic classes (forexample Oomycetes: Pythium and Phytophthora; Sterile fungi: Sclerotiumrolftsii and Rhizoctonia solani; Hyphomycetes: Fusarium spp. andAlternaria spp.) and a cytotoxic action on soil pathogens and parasites,in particular nematodes (in particular Meloidogyne incognita), insectsand weeds. Among the insects on which cytotoxic action is performed, thecytotoxic activity on wireworms is particularly interesting (for exampleAgriotes sordidus, Agriotes utulatus and Agriotes brevis). Thefungitoxic activity on fungi in the genus Fusarium is also particularlyinteresting.

It is important to point out that use of the flour permits exploitationof concentrations of GLs substantially impossible to achieve with thegreen manure technique.

Note that it is possible to use either flour of seeds of one singlevegetable variety, or a mixture of flour of seeds of a number ofvegetable varieties. In particular, the varieties used for their seedscan be chosen so as to create flour formulates with different modes ofaction, according to the need for persistence or volatility of thederivatives of the GLs and the required cytotoxic action. In fact, bothcharacteristics (volatility and cytotoxic action) can vary according tothe type of pathogen or parasite and the phase it is in at the time:vegetation (like mycelium) or conservation (sclerotium, oospore,chlamydospore or hibernating mycelium). Therefore, according to themodes of application (times and types of culture) of the targetpathogen(s) and/or parasite(s) and the need for improvement, thecompositions of the formulates can be varied. In this way it is alsopossible to obtain formulates for the control of telluric pathogens andparasites. Furthermore, it is possible to use the flour described abovealso in combination with other compounds, which in their turn can be ofeither synthetic or natural origin.

Further characteristics of the present invention will become clear fromthe description of some merely illustrative and non-restrictiveexamples. In particular, although the following examples refer to theuse of seed flour of Brassica carinata A.B, Eruca sativa M. and Barbareaverna as fungitoxic agents, the types of flour that can be used for saidpurpose are all those containing GLs and a glucosidasic orthioglucosidasic enzyme (in particular MYR). Furthermore, the flourcontaining GLs and a glucosidasic or thioglucosidasic enzyme (inparticular MYR) can be used also as a cytotoxic agent for other soilpathogens and parasites.

It is important to point out that the results illustrated in thefollowing examples can vary in quantitative but not qualitative terms,according to the vegetable varieties and ambient conditions in which theseeds are produced.

EXAMPLE 1

This example describes a method for obtaining seed flour of Brassicacarinata A.B, Eruca sativa M. and Barbarea verna.

Seeds of Brassica carinata A.B, Eruca sativa M. and Barbarea verna arecleaned and then ground separately with an ordinary rolling machineuntil a fine uniform flour is obtained. Each type of flour is separatelyde-oiled with n-hexane (Carlo Erba®) in a concentration of 1:10(weight/volume), leaving the suspension to be stirred in an agitator for12 hours at ambient temperature. Each type of flour is then separatelyfiltered on paper (Omniafiltra Supervelox filter, Cartiera del ToranoSpA, Naples), washed repeatedly again with n-hexane and placed in aventilated environment at ambient temperature for 24 hours in order toeliminate all remains of solvent.

EXAMPLE 2

This example describes an analysis method to determine the content ofGLs and the mirosinasic activity in seed flour.

Each type of flour, obtained as described in example 1, was analysed todetermine the content in GLs, using the procedure established by theregulation of the European Union 1864/90 for the analysis of rape seed(Official Journal of the European Communities, L 170, Mar. 7, 1990, p.0027-0034). The analysis was performed with HPLC technique (HighPerformance Liquid Chromatography), using a Hewlett Packard®Chromatograph Mod. 1090L with diode detector (λ=229 nm) and a column HPODS Hypersil C18, 5 μm 200×4.6 mm. The mobile phase consisted of amixture of water and acetonitrile with a gradient from 1% to 22% ofacetonitrile in 22 minutes and with a flow of 2 lm/min. The temperatureof the column was 35° C.

Furthermore the total myrosinase activity in the flour was alsoquantified via the pH-stat assay (Palmieri S., Iori R., Leoni, O.Comparison of Methods for Determining Myrosinase Activity, J. Agric.Food Chem. 1987, 35, 617-621). The analyses were repeated three times,giving the results in Table I, after calculation of the means andstandard deviations.

TABLE I Myrosinase GLs content activity Flour (μmoli g⁻¹ TQ) PrevalentGL (U g⁻¹) B. carinata 141.6 ± 4.8 Allyl GL 34.7 ± 0.4 (Sinigrine) E.sativa 161.1 ± 2.1 4-Methylthiobutyl 53.3 ± 4.2 GL (Glucoerucin) B.verna 143.7 ± 0.6 Fenyl-ethyl GL  2.4 ± 0.5 (Nasturtin)

The data given in Table I indicate that after cold grinding andextraction with n-hexane, the GLs and the enzyme MYR in active form arepresent in the flour. Therefore the de-oiled seed flour of P Brassicacarinata A.B, Eruca sativa M. and Barbarea verna contain the GLs-MYRsystem, and hence, in the presence of water, are able to producederivatives of GLs generated by enzymatic hydrolysis via MIR.

The analysis method described above can be used to identify thevegetable varieties in the seeds of which the GLs-MYR system is present.

EXAMPLE 3

This example describes a method for assessing the fungitoxic activity.

The fungitoxic activity test was performed starting from an isolate ofFusarium culmorum, isolated and identified as in Manici M. L. and CeratoC. (1992)—Studio su alcuni funghi agenti di marciume dei tuberi dipatata; Informatore Fitopatologico 9: 41-46, and references quoted init. This isolate is catalogued as fungo test at the micotech of theExperimental Institute for Industrial Cultivation (Bologna) and waschosen for the stability and regularity of growth that characterise it.

The test provided for assessment of the toxicity of the volatilecompounds released by the flour following hydration.

Doses of 0.01 g, 0.025 g, 0.05 g, 0.25 g and 1 g of the seed flour ofBrassica carinata, Eruca sativa and Barbarea verna analysed in example 2were tested separately. The control, included for assessment of thefungitoxic activity, consisted of pellets of commercial organic soilimprover based on fowl dung and torrefied leather.

The assay was based on assessment of the reduction of growth in coloniesof Fusarium culmorum in the presence of the products released by theflour following hydration, with respect to growth of the same fungus inthe presence of the control.

Colony discs of 4 mm taken from a one-week old colony of Fusariumculmorum were inoculated on a bottom of a Petri dish on an agarnutritive substrate (Potato Dextrose Agar) and immediately afteroverturned onto a bottom of a Petri dish containing the control and,separately, the flour in the different amounts (in a total volume ofapproximately 174 cm³) hydrated immediately beforehand. The two Petridish bottoms joined as above were sealed with parafilm, and theresulting dishes incubated for the time necessary for the colony on thecontrol to develop and invade at least ¾ of the dish. At this point thediameters of the colonies grown in the presence of the vapours releasedat the different doses of flour and control were measured.

The fungitoxic activity of the flour was expressed as inhibitioneffectiveness (EI) as a percentage with respect to the control, usingthe formula:EI(%)=[(Diameter of Control−Diameter of Treated Sample)/Diameter ofControl]×100.

Table II shows the results of the test.

The test was performed with three repetitions per treatment, thereforethe data shown in table II are the means of three values.

TABLE II Treatment Dose De-oiled flour (g/dish) EI % Eruca sativa 0.0181 0.025 100 0.05 100 0.25 100 1 100 Brassica carinata 0.01 72 0.025 1000.05 100 0.25 100 1 100 Barbarea verna 0.01 40 0.05 100 0.25 100 1 100The above data indicate that the products released by the seed flour ofBrassica carinata A.B., Eruca sativa M. and Barbarea verna, followinghydration, which activates the GLs-MYR system present in it, inhibit toa large extent growth of the fungus Fusarium culmorum.

EXAMPLE 4

This example describes a method for assessing the nematocide activity onnematodes, in particular root knot nematodes.

The nematocide activity test was performed with larvae of Meloidogyneincognita (Kofoid and White) Chitwoody, from soil in the Ferrara area.

The test was performed in jars with capacity of 100 g of soil to whichsecond age larvae had been added in order to assess the toxicity of thevolatile compounds released by the flour following hydration.

The doses equivalent to 20.10⁻², 40.10⁻² and 120.10⁻² Kg/m² of seedflour of Brassica carinata, Barbarea verna and Eruca sativa of example 2were tested separately. The control, included for assessment of thenematocide activity, consisted of non-treated soil and a fertiliser withtwo different organic compounds: fowl dung and torrefied leather(applied at the maximum test dose, i.e. 120.10⁻² Kg/M²).

The test was based on assessment of the mortality of the larvae ofMeloidogyne incognita in the presence of the flour and the non-biocideflour, with respect to the mortality in untreated soil.

The table shows the results of the test. The nematocide activity of theflour was expressed as the number of larvae remaining in the soil 10days after the treatment.

The test was performed with four repetitions per treatment, thereforethe data shown in table III are the means of four values.

TABLE III Dose N° larvae 100 g of Treatment Kg/m2 soil Blank — 14 Fowldung 40.10⁻² 8 Leather 40.10⁻² 5 B. Carinata 20.10⁻² 1 B. Carinata40.10⁻² 0 Eruca sativa 20.10⁻² 0 Eruca sativa 40.10⁻² 0 B. Verna 20.10⁻²2 B. Verna 40.10⁻² 2The above data indicate that the products released by the seed flour ofBrassica carinata A.B., Eruca sativa M. and B. Verna followinghydration, which activates the GLS-MYR system present in it, inhibit toa large extent the presence of Meloidogyne larvae in the soil.

EXAMPLE 5

This example describes a method for assessing insecticide activity, inparticular with regard to wireworms.

The insecticide activity test was performed with larvae of Agriotessordidus Illiger, Agriotes ustulatus Schäller, Agriotes litigiosus Rossiand Agriotes brevis Candeze, produced on breeding farms in Veneto.

The test was performed in jars with capacity of 1.4 l of soil to whichlarvae of wireworms of the above species at the 6^(th)-8^(th) stage ofdevelopment had been added in order to assess the toxicity of thevolatile compounds released by the flour following hydration.

The doses equivalent to 20 and 40 quintals ha⁻¹ of seed flour ofBrassica carinata and Eruca sativa of example 2 were tested separately.The control, included for assessment of the insecticide activity,consisted of untreated soil.

The test was based on assessment of the mortality of the larvae ofAgriotes in the presence of the flour which had undergone hydration,with respect to the mortality in untreated soil. Assessment of mortalitywas performed one hour after addition of the flour to the soil.

Table II shows the results of the test. The insecticide activity of theflour was expressed as inhibition effectiveness (EI) as a percentagewith respect to the control, using the formula:EI(%)=[(Mortality of Control−Mortality of Treated Sample)/Mortality ofControl]×100.

The test was performed with four repetitions per treatment, thereforethe data shown in table IV are the means of four values.

TABLE IV Treatment with de-oiled flour EI % Eruca sativa 92 Brassicacarinata 100

The above data indicate that the products released by the seed flour ofBrassica carinata A. B. and Eruca sativa M. following hydration, whichactivates the GLs-MIR system present in it, inhibit to a large extentthe presence of Agriotes larvae in the soil.

In particular, the following were observed:

-   -   a high insecticide action of the above-mentioned seed flour in        the space of only a few minutes;    -   a rapid disappearance of the active compounds (no insecticide        activity in the space of only a few days);    -   consequent low environmental impact.        No negative interactions were observed.

1. A method of killing soil pathogens and soil parasites comprising acontact step, during which at least one of a soil pathogen and a soilparasite is contacted with a composition comprising a seed flourcontaining at least one glucosinolate and the enzyme myrosinase, whereinthe seed flour contains a seed selected from the group consisting ofseeds of Brassica carinata A. B., seeds of Capparaceae, and seeds ofBarbarea verna; wherein the seed flour has been de-oiled at atemperature below 75° C.; and a release step, during which hydrolysis ofthe glucosinolate takes place in the presence of water so thatisothiocyanates are released from the composition to at least one of thesoil pathogen and soil parasite.
 2. The method of claim 1, wherein thesoil parasites are selected from the group consisting of nematodes andwireworms.
 3. The method of claim 1, wherein the soil parasites areselected from the group consisting of weeds and insects.
 4. The methodof claim 1, wherein the parasites are selected from the group consistingof Fusarium clumorum, Meloidogyne incognita (Kofoid and White)Chitwoody, Agriotes sordidus, Agriotes utulatus, and Agriotes brevis. 5.The method of claim 1, wherein the flour is fungitoxic to a fungi of thegenus Fusarium.
 6. A method of improving soil comprising a contact step,during which a soil is contacted with a seed flour containing at leastone glucosinolate and the enzyme myrosinase, wherein the seed flour istoxic to at least one organism selected from the group consisting of anematode, a wireworm, a weed, an insect, Meloidogyne incognita (Kofoidand White) Chitwoody, Agriotes sordidus, Agriotes utulatus, and Agriotesbrevis, and a fungi of the genus Fusarium; wherein the seed flour hasbeen de-oiled; and a release step, during which hydrolysis of theglucosinolate takes place in the presence of water so thatisothiocyanates are released from the composition to the organism. 7.The method of claim 6, wherein the seed flour has been de-oiled at atemperature below 75° C.
 8. The method of claim 6, and furthercomprising an identification phase to identify the seed flour containingat least one glucosinolate and at least one enzyme selected from thegroup consisting of glucosidasic enzymes and thioglucosidasic enzymes.9. The method of claim 8, wherein the at least one enzyme is myrosinase.10. A method of improving soil comprising a contact step, during whichthe soil is contacted with a flour made from at least one seed selectedfrom the group consisting of seeds of Brassica carinata A. B., seeds ofCapparaceae, and seeds of Barbarea verna wherein the flour has beende-oiled at a temperature below 75° C.; and a release step, during whichisothiocyanates are released from the composition into the soil.
 11. Themethod of claim 10, wherein the flour is toxic to at least one organismselected from the group consisting of a nematode, a wireworm, a weed, aninsect, Meloidogyne incognita (Kofoid and White) Chitwoody, Agriotessordidus, Agriotes utulatus, and Agriotes brevis, and a fungi of thegenus Fusarium.
 12. A method of improving soil comprising a contactstep, during which the soil is contacted with a composition comprising aseed flour containing at least one glucosinolate and enzyme myrosinase,wherein the seed flour contains a seed selected from the groupconsisting of seeds of Brassica carinata A.B., seeds of Capparaceae, andseeds of Barbarea verna; wherein the seed flour has been de-oiled; and arelease step, during which hydrolysis of the glucosinolate takes placein the presence of water so that isothiocyanates are released from thecomposition into the soil.
 13. The method of claim 12, wherein the seedflour has been de-oiled at a temperature below 75° C.
 14. The method ofclaim 13, wherein the seed flour has been de-oiled at ambienttemperature.
 15. The method of claim 12, wherein the seeds compriseseeds of Brassica carinata A.B.
 16. The method of claim 12, wherein theseeds comprise seeds of Capparaceae.
 17. The method of claim 12, whereinthe seeds are of at least two different vegetable varieties.
 18. Themethod of claim 1, wherein the seeds comprise seeds of Brassica carinataA.B.